GPR55 has recently been identified as a lysophosphatidylinositol (LPI)-sensitive receptor that may also mediate some off-target effects of cannabinoids. The broad central nervous system (CNS) distribution of GPR55 suggests its involvement in central physiology and pathology. Characterization of GPR55-/- (knock-out) mice reveals roles for the GPR55 receptor in inflammatory pain, neuropathic pain, and bone development while other studies indicate that GPR55 activation is pro-carcinogenic. Importantly, GPR55-/- mice show decreased inflammatory and neuropathic pain. Further, inhibition of LPI-induced activation of GPR55 may reduce neuropathic pain. The main goal of this application is to uncover the functional role of this new receptor, GPR55, in the periaqueductal gray (PAG), one of the most important regions involved in pain modulation and also a primary site of action of many analgesic compounds including cannabinoids. Our preliminary data revealed activation of GPR55 receptors in the PAG produces increases in intracellular calcium and cytoplasmic and mitochondrial reactive oxygen species in primary neurons and depolarization of PAG neurons in midbrain slice cultures. Furthermore, activation of GPR55 in PAG significantly reduced the pain threshold in rats. In other words, the activation of GPR55 in the PAG has a pro-nociceptive function. Thus, manipulating GPR55 signaling could be a new target for pain management. We propose to test a new hypothesis that the activation of GPR55 in the PAG has nociceptive response and antagonizing this receptor could have analgesic function. In the experiments under specific Aim 1, we will evaluate the GPR55-dependent Ca2+ response in PAG neurons. Studies under Aim 2 will use electrophysiology to characterize the membrane and synaptic activity responses of PAG neurons to GPR55 activation. In aim 3, we will determine the in vivo effect of GPR55 agonist(s)/antagonist(s) on PAG in several pain models. These studies will demonstrate the functional role of GPR55 in PAG, with the objective of identifying novel targets for effective therapeutic interventions. Specifically, we will determine whether the inhibition of GPR55 by antagonist(s) can be used as potential therapeutics for pain management.